Antibiotic compositions



United States Patent 3,269,908 ANTIBIOTIC COMPOSITIONS John E. Fahey, Gales Ferry, Conn., assignor to Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed July 5, 1963, Ser. No. 293,167 14 Claims. (Cl. 167-65) This invention relates to novel antibiotic compositions which comprise a tetracycline-type antibiotic together with an organic acid potentiating agent therefor. More particularly, it is concerned with oral compositions of the foregoing type which all contain a succinic acid derivative for the aforestated purpose, whereby there is thus achieved an enhancement of said antibiotic blood levels in the animal to whom they are so administered.

In this connection, it is to be understood that by the use of the term tetracycline-type antibiotic there is meant to be included not only such amphoteric tetracycline compounds as chlortetracycline, tetracycline, oxytetracycline, 6-deme-thylchlortetracycline, 6-deoxytetracycline, 6-demethyl-6-deoxytetracycline, 6-demethyl-6-deoXy-7-bromtetracycline, 6-demethyl-6-deoxy-6-methylene-5-oxytetracycline, etc., but also such acid addition salts thereof as the hydrochloride, hydrobromide, hydriodide, sulfate, nitrate, phosphate, acetate, lactate, maleate, citrate, tartrate and ascorbate, as well as base salts of same such as the sodium, potassium, calcium, quaternary ammonium or ethylene diamine salts thereof, or the aluminum gluconate complex or other organic acid complex salts thereof, or any other pharmaceutically acceptable salts of these particular antibiotics which happen to be therapeutically effective per se.

The succinic acid derivative of these novel compositions is an oxa, thia or imino diacid of the aforesaid type and is preferably one selected from the group consisting of 2,2- oxodisuccinic acid, 2,2'-thiooxodisuccinic acid and 2,2- iminodisuccinic acid, although the corresponding bis anhydrides, as well as the alkali metal and alkaline-earth metal salts thereof, in addition to the corresponding lower alkyl esters of these acids are also useful in this connection. 2,2'-oxodisuccinic acid is described and claimed by R. G. Berg in copending US. application Serial No. 255,- 167, filed January 31, 1963, now US. Patent No. 3,128,- 2-87, while the other two aforementioned organic acids are both old in the art. These compounds, i.e., the preferred acids, can all be represented by the following generic structural formula:

where X is a member chosen from the group consisting of oxygen, sulfur and imino. Certain other organic acids may also be employed in this connection in lieu of those mentioned above, such as structurally related compounds, including stereisomers thereof, but the aforementioned disuccinic acids are definitely preferred as previously discussed.

In .general, the amount of organic acid potentiating agent to be used with respect to the tetracycline component of the mixture may vary over a wide range, but it is usually more desirable in practice to employ at least 3,269,908 Patented August 30, 1966 a semi-equal amount by weight of the chosen acid and preferably an equal amount or excess of same since it is the cheaper of the two components. Optimum results, however, have been obtained with from about one-half to about thirteen parts by weight of the acid with respect to about one part by weight of the tetracycline component. As previously indicated, all these compositions give remarkably enhanced anti-biotic blood levels so that it is now within the realm of reality to achieve much higher tetracycline concentrations in the blood of the animals to whom they are so administered with a given amount of antibiotic compound than heretofore was ever thought possible or conversely, to achieve the same levels with less of the antibiotic than would normally be used alone for this same effect. The advantages afforded the use of this invention are therefore obvious. Moreover, the organic acid potentiating agent can be administered either before or after the administration of the antibiotic pro vided the time interval between the two is not too excessive, i.e., not more than about 10-20 minutes. However, it is preferred in practice to use the two main components of the mixture together (i.e., at the same time) as will hereinafter be discussed.

A preferred unit dosage form for administering these novel compositions is most conveniently a powdered mixture of the two principal components encased in a soft or hard-shelled .gelatin capsule. This dosage form may contain from about 25 mg. up to about 500 mg. of the desired tetracycline together with from about one-half to about thirteen parts by weight of the chosen organic acid. An inert diluent, such as a pharmaceuticallyacceptable carrier like starch, lactose or milk sugar, or glucosamine, may also be present if so desired. Conversely, it is also possible to administer these compositions in granulated form as such or else compressed into tablets for oral administration, as well as in the form of aqueous suspensions, elixirs, lozenges, troches, candies and pediatric drops, etc. When administered parenterally, they can be given to the animal in the form of an aqueous dispersion or solution for intravenous injection, or as an organic solution or suspension for intramuscular injection.

Moreover, these compositions may also be administered to domesticated animals, such as cats, dogs, sheep, chickens, turkeys, ducks, hogs, rabbits, and the like, by way of their feeds or drinking water in addition to the direct route previously mentioned. For instance, the oral unit dosage forms previously discussed may be added as such to animal feeds or, and preferably, the novel compositions themselves may be blended into powders for incorporation directly into the feeds. The antibiotic component of the 'feed may vary anywhere from about 50 g. up to about 1000 g. per ton of feed, while the organic acid potentiating agent must be present to the extent that it is at least about 0.1% by weight based on the total weight of the composition. Needless to say, the tetracyclinetype antibiotic component and the organic acid potentiating agent component may either be administered via one component of the animal's feed or else they may be uniformly blended or distributed therein throughout a completely-mixed feed. In this latter connection, it is to be noted that a wide variety of different feed components may be of use in the nutritional diets called for. Thus, the proper feed compositions should normally contain nutritionally-balanced quantities of carbohydrate, fat and protein, together with the usual vitamin and mineral supplementary sources.

When the novel compositions of this invention are to be used in feeds, a particular type of conventional feed material for just such purposes is one which generally follows the suggested formulae set forth on page six of the authoritative pamphlet issued by the National Research Council of Washington, D.C., on June 1944 and entitled Recommended Nutrient Allowances for Poultry. For instance, feed compositions are recommended to contain roughly from between about 50% and about 80% of grains, such as ground grain or grain by-products, molasses and other sugar by-products; from between about 3% and about 10% of animal protein, such as fish meal, meatscraps, and the like; from between about and about 30% of vegetable protein, such as soybean oil meal, peanut meal, cottonseed meal, and the like; and from between about 2% and about 5% of minerals, such as bone meal, limestone, and so forth; together with supplementary vitaminaceous sources, such as thiamine, riboflavin, niacin, etc. Furthermore, the compositions of this inevntion may either be used in said feeds alone or in conjunction with various other antibiotics and chemotherapeutic agents, such as penicillin, bacitracin, sulfaquinoxaline, phenylarsonic acid, hydroxyzine, oleandomycin, and so forth.

In accordance with a more specificembodiment of this invention, a typical feedstufi for poultry may be prepared by mixing the following ingredients together in the 1 The trace mineral mix constitutes 0.05% by weight of the feed and the ingredients contained therein are as follows together with their weight percentages in the premix: manganese, 24% iodine, 0.48% iron, 8.0% copper, 0.8% zinc, 0.4% and cobalt, 0.8%.

The Vitamin premix constitutes 0.55% by weight of the feed and the ingredients contained therein are listed below as follows together with their percent concentrations in the premix:

Percent Vitamin A (10,000 I.U./g.) 10.0 Vitamin D3 (3,000 I.C.U-/g 10.0 Choline chloride (25%) 35.25 Nlacin (80%) 0.55 til-Calcium pantothenate (45%) 10.0 Riboflavin (4 g./lb.) 0.39 Vitamin B12 (60 mg./1b.) 0.20 V1gofac 3 30.0 Fine corn meal 3.75

Vigofac is the registered trademark name of the Chas. Pfizer & 00., Inc. for unidentified poultry-livestock growth factors obtained from streptomyces fermentatron sources.

Needless to say, many other comparable feeds mayalso be blended in a similar manner, such as, for example, those which contain lesser amounts of calcium, for instance, up to about 0.5% by weight of calcium as compared to the 1.2% calcium concentration level which is contained in the feeds of the foregoing description.

This invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contr iry, it is to be clearly understood that resort may be had to various other embodiments, modifications and equivalents thereof which readily suggest themselves to those skilled in the art without departing from the spirit of the present invention and/ or the scope of the appended claims.

EXAMPLE I Ten eight-week-old chickens (five male and five female) of the Vantress-White Rock Cross broiler variety were each fed a standard animal feedstuif diet of the Average Oxytetra- Potentiation cycline Blood Sera Effect (Index) Substance Levels (meg/ml.)

24 hrs. 72 hrs. 24 hrs. 72 hrs.

Oxytetracycline H01 0. 2280 0.2140 100 Oxytetracycline H01 plus Oxa acid 0. 2286 0. 2079 103 From the data presented in the above table, it is obvious that 2,2-oxodisuocinric acid produced increases in oxytetracycline blood sera levels over that of the controls during assay periods.

EXAMPLE II Thirty-five (35) eleven-week-old female Vantress- White Rock Cross broilers were divided into five pens of seven birds each. These birds were fed the same standard feed diet as described before and given water continuously throughout the entire test period. One group received a slug dose i.e., an oral dose, of oxytetracycline hydrochloride at the 250 mg. level together with onehalf that amount by weight of 2,2'-oxodisuccinic acid, while another group received the same two chemicals in a 1:2 weight ratio, respectively, i.e., 250 mg. oxyeteracycline hydrochloride and 500 mg. 2,2 oxodisuccinic acid. This was then repeated with another group of animals using citric acid in place of the oxa acid at the same two dose levels for comparison purposes (again, a total of 14 animals or seven per group), while a further group served as the controls and were given'the antibiotic alone (250 mg. oxytetracycline hydrochloride) without the organic acid. At the end of two :and six hours after the initial slug dosing, oxytetracycline blood sera levels were determined by standard assay procedures and the relsaults obtained are presented below in the following ta le:

From the data in the above table, it is obvious that 2,2'-oxodisuccinic acid gives much greater blood levels with oxytetracycline than does citric acid in addition to bring significantly higher than the controls.

EXAMPLE III The procedure described in Example II. is followed except that 2,2'-thio-oxodisuccinic acid is employed in place of the oxa acid used therein on the same Weight basis. In like manner, 2,2'-iminodisuccinic acid is also used. In both these cases, the results obtained are substantially identical with those reported in Example II for the oxa acid,

EXAMPLE IV The procedure described in Example II is followed except that chlortetr-acycline is employed in place of oxytetracycline, on the same weight basis, as the antibiotic of choice. In like manner, tetracycline hydrochloride is also employed. In both these cases, comparable results with respect to oxytetracycline are obtained.

EXAMPLE V The procedure described in Example II was repeated with fifty-four (54) twelve-week-old female Vantress White Rock Cross broilers divided into groups of six birds each. In this case, the oxytetracycline hydrochloride was employed at the 25 mg. slug dose level and the 2,2-oxodisuccinic acid (oxa acid) was employed at various levels ranging from 80 mg. to 320 mg. in either 5 ml. or ml, amounts (of water). The results obtained in this manner are presented below in the following table, which also includes similar information obtained with citric acid at 320 mg. for comparison purposes:

Average Oxytetra- Potentiation cycline Effect Dose Treatment Blood Sera (Index) Potentiator (mg) Volume Assay (1111.) (meg/ml.)

2 hrs. 6 hrs. 2 hrs. 6 hrs.

5. 0 0.322 0. 119 100 100 5. 0 0. 478 0. 259 148 218 10.0 0.299 0. 148 98 124 5. O 0. 397 0. 220 123 184 10. 0 0. 591 0. 17 0 184 143 5. 0 0. 544 0.219 169 184 Do 320 10. 0 0. 740 0. 263 230 221 Citric acid 320 5. 0 0. 289 0. 156 90 131 Do 320 10. 0 0. 325 0. 143 101 120 From the data in the above table, it is obvious that 2,2- oxodisuccinic acid (oxa acid) is an eifective potentiating agent for oxytetracycline when administered in slug doses and that approximately one-fourth the quantity of this compound with respect to the amount of citric acid employed produced essentially the same results at the end of the 6-hour period.

EXAMPLE VI The procedure described in Example V is followed except that 500 mg. of oxytetracycline hydrochloride is employed instead of 25 mg. In like manner, tetracycline hydrochloride and chlortetracycline hydrochloride are each similarly employed at both these same dose levels, i.e., at 25 mg. and 500 mg. respectively. In all these cases, the results obtained are substantially the same with respect to degree of potentiation as those previously reported in Example V. Comparable results are also obtained in all these cases when 2,2'-thiooxodisuccinic acid and 2,2'-iminodisuccinic acid are each individually employed in place of the oxa acid previously used.

EXAMPLE VII Seventy (70) six-week-old chickens of the Vantress- White Rock Cross broiler variety were divided into seven employed in lieu thereof. The results obtained in this manner are summarized below in the following table:

Average Oxytetra- Potentiating Effect; Concn. cyeline Blood Sera (Index) Potentiating Level, Levels (meg/ml.)

Compound percent 24 hrs. 72 hrs. 24 hrs. 72 hrs.

None 0. 209 0. 221 100 Oxa acid. 0.01 0.188 0.245 90. 1 111 0. 1 0.223 O. 240 106. 6 108. 8

From the above table, it is obvious that 2,2'-oxodisuccinic acid (oxa acid) achieved its greatest potentiating effect at the 2.0% concentration level and that 2,2-thiooxodisuccinic acid (thia acid) is quite similar to the aforementioned oxa acid with respect to its potentiating effect at the two different dose levels at which it was tested,

In like manner, 2,2'-iminodisuccinic acid proved to be equally eifective as a potentiating agent as the thia acid when tested at the same two dose levels as indicated above.

EXAMPLE VIII A total of sixteen fasting (for about 15 hrs.) dogs, each weighing anywhere from about 15 to 49 lbs., were used in this experiment. Seven of the dogs received the standard human capsule of 250 mg. of oxytetracycline hydrochloride with glucosamine, while the remaining nine each received the experimental capsule that contained 250mg. of oxytetracycline hydrochloride together with 250 mg. of 2,2'-oxodisuccinic acid. At the end of three and six hours after the capsule administration, blood samples for serum assay were obtained from the dogs. The results obtained in this manner are presented below in the following table:

Sixty-four (64) eight-week-old chickens of the Vantress- White Rock Cross broiler variety were divided into groups of four male and four female birds each. They were then each fed the standard basal animal feedstuif ration without added antibiotic and subjected to the following treatment, employing oxytetracycline hydrochloride (250 mg.) and organic acid potentiating agent at the two dose levels indicated, and including both water solution and capsule form for the 2,2'-oxodisuccinic acid (oxa acid).

Average Oxytetracycline Potentlating Blood Sera Levels (mcg./ Potentiatmg Dose (mg./ Dosage ml.)

Agent ml.) Form 3 hrs. 6 hrs.

Water- 1. 817 (100) 1. 423(100) Capsule 1. 717(100) 0. 491 (10 Water..- 6. 333(34 4. 258 (299) Capsule 5. 262(306) 2. 025 (412) Water..- 10. 233(568) 4. 717(331) Capsule 12. 900(751) 4.150(845) d0 2. 592(151) 1. 656 (337) Imlno acld 250 do 6. 238(363) 1. 950 (397) From the data presented in the above table (where the potentiation indices are given in parentheses), it can be calculated that average potentiating effects of 329 and 787 were obtained with 250 mg. and 500 mg. capsule doses of 2,2'-oxodisuccinic acid (oxa acid), respectively, as com- .pared to corresponding values of 231 and 453 for the aqueous solutions containing same. The analogs of 2,2- oxodisuccinic acid, viz., 2,2'-thiooxodisuccinic acid (thia acid) and 2,2'-iminodisuccinic acid (imino acid), also produced marked potentiating effects.

EXAMPLE X Sixty (60) eight-weekcld chickens of the Vantress- White Rock Cross broiler variety were divided into six 'groups of five male and five female birds each and fed the standard ration used before. One set of three such groups received oxytetracycline hydrochloride at levels of '200, 500, and 1000 grams per ton, respectively, together with 2,2'-oxodisuccinic acid (oxa acid) at the 2.0% concentration level. The other set of birds received the antibiotic alone, without the added acid, at the same three dose :levels previously indicated. At the end of 24, 48 and 72 hours after initiation of the feeding, the birds were bled by heart puncture in order to run blood sera assays on the resulting samples. this manner are summarized below in the following table:

The results obtained in two compounds at the end of the aforementioned time periods:

Average Antibiotic Oxa Acid Potentiation Indices Antibiotic Concn. Level,

percent 24 hrs. 72 hrs.

None 100 100 2 241 198. 5 None 100 100 From the data presented in the above table, it is obvious that 2,2-oxodisuccinic acid produced a potentiation of the oxytetracycline and chlortetracycline blood levels.

EXAMPLE XIII A group of five albino rats received an initial dose of 6-demethyl-6-deoxy-6-methylene-5-oxytetracycline hydrochloride (GS-2876) orally via an oral tube at the 125 mg./kg. level. A second group received the same dose together with an equal amount of 2,2'-oxodisuccinic acid (125 mg./kg.). A third group received no antibiotic nor oxa acid whatsoever and served as the controls, while a fourth group received the oxa acid alone. The animals were then placed in metabolism cages for 18 hours post From the above table, it is obvious that 2,2'-oxodisuccinic acid (oxa acid) at the 2% concentration level potentiates oxytetracycline at all three dose levels tested.

EXAMPLE XI The procedure described in Example X is repeated except that oxytetracycline hydrochloride is now employed at concentration levels of 50, 100 and 150 grams per ton, respectively. In each and every case, the results obtained are comparable in order of magnitude to those previously reported in the foregoing example for the higher concentration values.

EXAMPLE XII Forty-eight (48) young chickens of the Vantress- White Rock Cross broiler variety were placed into pens of six male and six female birds each. The animals were then fed the standard animal basal feedstuif ration, with one group (12 animals) receiving oxytetracycline at the 400 g. per ton level together with 2,2'-oxodisuccinic acid in the amount indicated below, while another group served as the controls and received no such oxa acid. In like manner, these tests were again repeated with two other groups of animals only employing chlortetracycline in place of the oxytetracycline. At the end of 24 and 72 hours after initiation of said treatment, the birds were bled in order to determine blood sera levels on their samples. The results obtained in this manner are summarized below in the following table, which indicates the average antibiotic potentiation indices for each of these dose and the total volume of urine excreted was collected. Food was withheld from the animals throughout this entire test period, but water was given them ad libitum. The results obtained in this manner are shown below in the following table:

Oral Dose Average Percent Substance (mg/kg.) Dose Excreted (percent) Nona No activity Oxa Acid N 0 activity GS-2876 125 2. 28 GS-2876 plus Oxa aci 66 From the data presented in the above table, it can be seen that 2,2-oxodisuccinic acid (oxa acid) at the 125 mg./kg. level gives much greater uninary excretion levels (about six times as much) with 6-demethyl-6-deoxy-6- methylene-6-oxytetracycline hydrochloride than does the antibiotic itself when used alone.

What is claimed is:

1. An antibiotic composition comprising a tetracyclinetype antibiotic and a compound selected from the group consisting of 2,2'-oxodisuccinic acid, 2,2'-thiooxodisuccinic acid and 2,2' iminodisuccinic acid.

2. A composition as claimed in claim 1 wherein the tetracycline-type antibiotic is oxytetracycline.

3. A composition as claimed in claim 1 wherein the e r y lin -type antibiotic is tetracycline.

4. A composition as claimed in claim 1 wherein'the tetracycline-type antibiotic is chlortetracycline.

5. An antibiotic composition as claimed in claim 1 wherein the amount of organic acid present by weight is at least equal to one-half that of the tetracycline antibiotic.

6. An antibiotic composition is unit dosage form suit able for oral administration comprising from about 25 mg. up to about 500 mg. of a tetracycline-type antibiotic and from about one-half to about thirteen parts by weight of a compound selected from the group consisting of 2,2- oxodisuccinic acid, 2,2'-thiooxodisuccinic acid and 2,2- iminodisuccinic acid with respect to the tetracycline antibiotic.

7. A composition as claimed in claim 6 wherein the organic acid is 2,2-oxodisuccinic acid.

8. A composition 'as claimed in claim 6 wherein the organic acid is 2,2-thiooxodisuccinic acid.

9. A composition as claimed in claim 6 wherein the organic acid is 2,2'-iminodisuccinic acid.

10. An animal feed composition comprising a nutritionally-balanced animal feed to which there is added a tetracycline-type antibiotic and a compound selected from the group consisting of 2,2-oxodisuccinic acid, 2,2'-thiooxodisuccinic acid and 2,2-iminodisuccinic acid.

11. A feed composition as claimed in claim 10 wherein the antibiotic is present at a concentration level that is in the range of from about 50 g. up to about 1000 g. per ton of feed.

12. A feed composition as claimed in claim 10 wherein the organic acid member is present to the extent of at least about 0.1% by Weight based on the total weight of the composition.

13. A method of potentiating the blood levels of a tetracycline-type antibiotic when orally administered, which comprises the step of orally administering the tetracyclinetype antibiotic in conjunction with a compound selected from the group consisting of 2,2'-oxodisuccinic acid, 2,2- thiooxodisuccinic acid and 2,2'-iminodisuccinic acid.

14. A method as claimed in claim 13 wherein the amount of organic acid employed by weight is at least equal to one-half that of the tetracycline-type antibiotic.

Eisner et al.: The Enhancement of Serum Levels of Aureomycin in Experimental Animals, J. Pharm. EXptl. Therap. 108 (4): 442-449, August 1953.

LEWIS GOTTS, Primary Examiner.

S. K. ROSE, Assistant Examiner. 

1. AN ANTIBIOTIC COMPOSITION COMPRISING A TETRACYCLINETYPE ANTIBIOTIC AND A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 2,2''-OXODISUCCINIC ACID, 2,2''-THIOOXODISUCCINIC ACID AND 2,2''-ININODISUCCINIC ACID. 